DESCRIPTION (taken from the application) This research proposal addresses the question of which genes of Helicobacter pylori are required for colonization in vivo, an essential step leading to gastritis, ulcer, and stomach malignancies. Several essential proteins in H. pylori have already been identified by analysis of individual genes. The proposal has two major goals. The first is the characterization of Hpn, a newly identified protein present in H. pylori, H. mustelae and H. felis. We report preliminary evidence that hpn mutants are unable to colonize a rodent model, and this will be verified by an in vivo competition assay using wild type and hpn-mutants. Hpn is a metal binding protein, and experiments are proposed to correlate Hpn expression and protection from the toxic effects of bismuth in vivo. Hpn:GFP fusions show unipolar localization in the cell and confirmation of this asymmetry may help define Hpn function. In our second goal we will take three separate genetic approaches to identify genes or groups of genes important in the biology of H. pylori, and its ability to colonize the gastric mucosa. To date, no general approach to finding such genes has been carried out in H. pylori. The first approach has involved the construction of a novel urease reporter system to identify acid-inducible genes in vitro, and identification of genes whose expression is induced to enable colonization of a rodent model. Our second approach is a signature-tagged transposon mutagenesis method. This will identify genes that are essential for colonization in vivo. And third, we will use a novel Cre-lox reporter system, which will select for genes specifically expressed in vivo. The Cre-lox system will identify a much larger set of genes important for survival, and will include genes involved in regulatory, metabolic and pathogenic processes. Each system is designed to identify different functional groups of genes, and overlap is not only expected, but desirable.